Trimethylguanosine nucleoside inhibits cross-linking between Snurportin 1 and m3G-CAPPED U1 snRNA

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin alpha/beta. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import.     

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.     

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m₃G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m₃G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m₃G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA.     

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein,survival of motor neuron

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.     

Cross-talk between snurportin1 subdomains

The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-beta. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-beta, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-beta binding domain (IBB) disrupted the interaction with importin-beta, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-alpha.     

Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways

The inhibitory effects of wheat germ agglutinin and mAb 414 on the nuclear import of all types of U snRNAs indicate that they cross the nuclear envelope through the nuclear pore complex. However, the import of different U snRNAs occurs by kinetically distinct targeting pathways that can be distinguished from one another by the competitive effects of free trimethylguanosine cap dinucleotide (m3GpppG) and P(Lys)-BSA, an efficient synthetic karyophile based on the nuclear localization signal of SV40 large T antigen. The import of U snRNAs that contain 5' m3GpppN caps and are complexed by Sm proteins (U1, U2, U4, and U5) is competed by coinjection with free m3GpppG, indicating a shared transport factor, but not by P(Lys)-BSA. The import of U6 snRNA, which lacks a m3GpppN cap and is not complexed by the Sm proteins, is competed by P(Lys)-BSA but not by free m3GpppG. Thus, by the criterion of kinetic competition, U6 snRNA import is identical to that of the karyophilic proteins P(Lys)-BSA and nucleoplasmin. Uniquely, the import of U3 snRNA, which contains a m3GpppN cap but does not bind Sm proteins is not competed by either free m3GpppG or P(Lys)-BSA. Thus, U3 snRNA appears to be imported by a novel third kinetic pathway.     

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.     

5′ and 3′ end modifications of spliceosomal RNAs in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

5′ caps provide recognition sequences for the nuclear import of snRNAs. The 5′ and 3′ ends of snRNAs were studied in Plasmodium falciparum with a modified adapter ligation method, which showed that 5′ ends of U1, U2, U4, U5 and U6 snRNAs are capped. In P. falciparum, the 3′ ends of U1, U2, U4 and U5 snRNAs have free hydroxyl groups whereas U6 snRNA has a blocked 3′ end. An immunoprecipitation assay for trimethyl guanosine caps shows that the cap structures of parasite U1-U5 snRNAs are hypermethylated while U6 snRNA may be γ-mono-methylated. Bioinformatics analysis of proteins involved in hypermethylation and trafficking of snRNAs indicates that the methyltransferase TGS1 is present in the P. falciparum genome. PfTGS1 is larger than its orthologs and may have transmembrane domains in the C-terminus. Surprisingly, the snRNA trafficking protein Snurportin is absent from the P. falciparum genome suggesting that reminiscent of yeast, parasite snRNAs may be retained in the nucleus.     

Assembly and intracellular transport of snRNP particles.

The assembly of the major small nuclear ribonucleoprotein (snRNP) particles begins in the cytoplasm where large pools of common core proteins are preassembled in several RNA-free intermediate particles. Newly synthesized snRNAs transiently enter the cytoplasm and complex with core particles to form pre-snRNP particles. Subsequently, the cap structure at the 5' end of the snRNA is hypermethylated. The resulting trimethylguanosine (TMG) cap is an integral part of the nuclear localization signal for snRNP particles and the pre-snRNP particles are rapidly transported into the nucleus. SnRNP particles mature when snRNA-specific proteins complex with the particles, in some cases, just before or during nuclear transport, but in most instances after the particles are in the nucleus. In addition, U6 snRNA hybridizes with U4 snRNA to form a U4/U6 snRNP in the nucleus. The transport signals are retained on the snRNP particles and proteins since existing particles and proteins enter the reformed nucleus after mitosis.     

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.     

5′-terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices : Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera

Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.     

CRM1-mediated recycling of snurportin 1 to the cytoplasm

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.     

Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA.

A class of RNA-containing particles, U small nuclear/nucleolar ribonucleoprotein particles (U snRNP), are well known to be targets for sera from patients with various autoimmune diseases. In the most cases the protein components carry the antigenic determinants. We have identified serum autoantibodies from three patients with systemic sclerosis that were directed against U1-U5 snRNA by immunoprecipitation of deproteinized 32PO4 labeled HeLa cell total RNA. By competitive radioimmunoprecipitation assays, an experimentally induced anti-2,2,7-trimethylguanosine (TMG) cap structure mAb inhibited the reaction of these antisera. In addition, IgG isolated from the antisera inhibited the anti-TMG mAb reaction to the U snRNA. Furthermore, a structural analog, 7-methylguanosine-triphosphate, competitively inhibited the reaction of the antisera to the U snRNA. Thus we concluded that the TMG cap structure of the U snRNA could be a target for serum autoantibodies.     

The ribonucleotidyl transferase USIP-1 acts with SART3 to promote U6 snRNA recycling

The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1–U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling.     

In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA

In this paper we describe a method for preparing native, RNA-free, proteins from anti-m₃G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B, D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm³. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.Abbreviations PEG Polyethelene glycol - PMSF Phenylmethyl sulfonylfluoride - TP total proteins - mAb monoclonal antibody